Composition, structure and interactions of the Cleavage and Polyadenylation Specificity Factor (CPSF) involved in mammalian mRNA 3' end processing

真核生物mRNAs的末端是由一个加工反应产生的，在这个过程中，mRNA前体首先通过内切作用缩短，然后上游的切割片段被多聚腺苷化。在加工反应所需的大量蛋白质中，异寡聚体切割和多腺苷基化特异性因子(CPSF)被认为是核心因素：CPSF既参与切割又参与多腺化，它包含催化前mRNA切割的酶，它识别AAUAAA多腺苷信号，并赋予Poly(A)聚合酶对含有AAUAAA的底物RNA的特异性。然而，CPSF的组成和分子间的相互作用还不是很清楚，我们已经从六个高表达的亚基中重组了CPSF，重组因子在多聚腺苷化中具有活性。六个亚基中的两个对于这个反应来说是不必要的，但对于前mRNA的切割可能是必不可少的。由于多聚腺苷酸化所需的另外两种蛋白质--聚(A)聚合酶和核多聚(A)结合蛋白可以在大肠杆菌中产生，现在可以进行完全重组的反应。在AAUAAA依赖的多腺化作用中，最小的CPSF活性的组成将根据亚基的类型及其结合的化学计量来确定。将研究CPSF的RNA结合特异性和单个亚基对RNA识别的贡献。这涉及AAUAAA多聚腺苷信号和可能被CPSF识别的附加序列。CPSF亚基之间的相互作用以及最重要的是与聚(A)聚合酶的相互作用将被确定。我们将询问与Poly(A)聚合酶的相互作用是否受Poly(A)尾巴长度的调节，正如已发表的Poly(A)尾长控制模型所假设的那样。我们将研究CPSF是如何刺激聚(A)聚合酶的，以及另外两种加工因子，切割因子I和切割刺激因子，两者都可以以过量生产和纯化的形式提供，是否真的可以像报道的较少纯化的系统那样，在重组系统中刺激多聚腺苷基化。Symplekin和切割因子II将过度表达，以完成已知的3个主要加工因子的集合，并尝试从过度生产和纯化的蛋白质中重建切割反应。最后，我们将与结构生物学家合作，通过低温电子显微镜和X射线结晶学的手段，帮助阐明CPSF的结构

The ends of eukaryotic mRNAs are generated by a processing reaction in which the mRNA precursor is first shortened by endonucleolytic cleavage and the upstream cleavage fragment is then polyadenylated. Within a large assembly of proteins required for the processing reaction, the heterooligomeric cleavage and polyadenylation specificity factor (CPSF) is considered the central factor: CPSF is involved both in cleavage and polyadenylation, it contains the enzyme catalyzing pre-mRNA cleavage, it recognizes the AAUAAA polyadenylation signal, and it endows poly(A) polymerase with specificity for an AAUAAA-containing substrate RNA. However, neither the composition of CPSF nor its molecular interactions are well understood.We have reconstituted CPSF from six overexpressed subunits.The reconstituted factor is active in polyadenylation. Two of the six subunits are dispensable for this reaction, but are probably essential for pre-mRNA cleavage. As the two other proteins required for polyadenylation, poly(A) polymerase and the nuclear poly(A) binding protein, can be produced in E. coli, a fully reconstituted reaction is now available for mechanistic studies.The composition of the minimal CPSF active in AAUAAA-dependent polyadenylation will be determined with respect to types of subunits and the stoichiometry of their association.The RNA binding specificity of CPSF and the contributions of individual subunits to RNA recognition will be studied. This concerns both the AAUAAA polyadenylation signal and additional sequences possibly recognized by CPSF.The interactions of CPSF subunits with each other and, most importantly, with poly(A) polymerase will be determined. We will ask whether the interaction with poly(A) polymerase is regulated by the length of the poly(A) tail, as postulated in a published model for poly(A) tail length control. We will investigate how CPSF stimulates poly(A) polymerase and whether two other processing factors, cleavage factor I and cleavage stimulation factor, both available in overproduced and purified form, can indeed stimulate polyadenylation in a reconstituted system as has been reported for a less purified system.Symplekin and cleavage factor II will be overexpressed to complete the set of known 3 prime processing factors, and a reconstitution of the cleavage reaction from overproduced and purified proteins will be attempted. If this is not successful, identification of the missing factor(s) will be attempted.Finally, we will collaborate with structural biologists to help elucidate the structure of CPSF by means of cryo-electron microscopy and X-ray crystallography